Cyclin-Dependent Kinase 6 Phosphorylates NF-κB P65 at Serine 536 and Contributes to the Regulation of Inflammatory Gene Expression
Figure 5
Phosphorylation of Ser536 of p65 by CDK6/cyclin complexes contributes to activation of NF-κB-dependent transcription.
(A) HEK293IL-1R cells were transiently transfected with the indicated amounts (µg) of expression vectors and a NF-κB-dependent luciferase gene. After 24 h cells were lyzed and luciferase activity was normalized for co-transfected SV40-promoter driven ß-galactosidase. The graph shows the mean luciferase activity +/− s.e.m. from three independent experiments performed in duplicates relative to the vector control. Expression of cotransfected MYC-cyclin D1, v-cyclin derived from Kaposi’s sarcoma herpesvirus (MYC-v-cyclin) and HA-CDK6 was analyzed by immunoblotting using anti MYC or anti CDK6 antibodies for all experiments in parallel. One representative blot is shown. (B) NF-κB p65-deficient MEFs stably reconstituted with wild type p65 (−/−/wt) or the Ser536A mutant (−/−/Ser536A) as described [64] were transiently transfected with empty vector or HA-CDK6 plus v-cyclin expression vectors. Cotransfected luciferase reporter genes were either controlled by three NF-κB binding sites (upper graph) or alternatively by the cyclin D1 promoter (lower graph). After 24 h cells were lyzed, luciferase activity was determined and normalized for co-transfected SV40-promoter driven ß-galactosidase activity. Shown is the mean luc. activity +/− s.e.m. from three (lower graph) or four (upper graph) independent experiments performed in duplicates relative to the vector control. The lower panel shows a representative immunoblot for the detection of p65 wt and Ser536Ala mutant, p65 Ser536 phosphorylation and ectopically expressed CDK6. Asterisks indicate significant changes of reporter gene activity.